Prostate cancer is the most common solid tumor and the second leading cause of cancer deaths in US men, with nearly 200,000 newly diagnosed cases per year and 30,000 deaths per year. Despite advances in prevention and early detection using Prostate Specific Antigen (PSA), nearly 20% of newly diagnosed patients present with metastatic disease, which constitutes the majority of prostate cancer mortality. No specific imaging or effective treatment is yet available for these patients with disseminated disease. Prostate-specific gene-expression imaging and therapy are promising technologies with powerful clinical applications. Due to inefficient gene transduction in vivo, prostate-specific promoter activities need to achieve magnitudes similar to those of ubiquitously active viral enhancers such as Simian Virus 40 (SV40) and Cytomegalovirus (CMV). The systematic improvement of the PSA regulatory regions we undertook resulted in the development of a TSTA (two-step transcriptional activation) system, which exhibits activities 2- and 800-fold higher than CMV and the native PSA promoter, respectively, and still retains a significant degree of prostate selectivity and androgen inducibility. The key objectives of this proposal is to further refine the vectorology by creating the most optimal combinations of the TSTA system and demonstrate its activity and specificity by non-invasive imaging in living animals. The vigorous evaluation of in vivo expression of the TSTA system will be translated to the development of pre-clinical therapeutic paradigms that couples tumor cell eradication to imaging. Specifically, we will create a single most advanced "gutless" adenoviral vector capable of simultaneously expressing an imaging gene, the cytotoxic herpes simplex virus thymidine kinase gene and an anti-angiogenic TSP-1 gene all regulated by the potent prostate-specific TSTA system. The ultimate application of this technology is to administer this multi-modal targeted vector systemically in order to detect and localize metastases, which will be followed by the activation of gene-based cytotoxic therapy. Validation of this potent yet specific approach in relevant prostate cancer animal models will ensure safety and efficacy in future clinical applications. [unreadable] [unreadable]